Changing the lactose permease of Escherichia coli into a galactose-specific symporter
AUTOR(ES)
Guan, Lan
FONTE
The National Academy of Sciences
RESUMO
N-ethylmaleimide (NEM) modification of a lactose permease mutant containing a single-Cys in place of Ala-122 (helix IV) abolishes active lactose transport. Moreover, lactose, melibiose, and β,d-galactopyranosyl 1-thio-β,d-galactopyranoside protect against NEM inactivation of lactose transport and/or alkylation of Cys-122 by [14C]NEM. Remarkably, however, d-galactose transport is relatively unaffected by NEM, and the monosaccharide affords no protection against NEM inactivation of lactose transport. Consistently, competitive inhibition of [14C]galactose transport by lactose, melibiose, or β,d-galactopyranosyl 1-thio-β,d-galactopyranoside is drastically reduced after NEM modification, whereas inhibition by unlabeled galactose is unaffected. The results indicate that alkylation of Cys-122 selectively inhibits binding and transport of disaccharides, whereas transport of the monosaccharide galactose remains largely unaffected. In addition, although the conservative mutation Ala-122 → Ser causes only mild inhibition of lactose transport, the mutations Ala-122 → Phe and Ala-122 → Tyr lead to marked inhibition. In contradistinction, none of these replacements has a marked effect on galactose transport. The results demonstrate that Ala-122 is a component of the ligand-binding site and provide a strong indication that the side chain at position 122 abuts on the nongalactosyl moiety of d-galactopyranosides. This is in contrast to Cys-148, a neighboring residue in helix V, that interacts with the hydrophobic face of the galactosyl moiety of d-galactopyranosides.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=124451Documentos Relacionados
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